scholarly journals Entry of Neisseria meningitidis into Mammalian Cells Requires the Src Family Protein Tyrosine Kinases

2010 ◽  
Vol 78 (5) ◽  
pp. 1905-1914 ◽  
Author(s):  
Heiko Slanina ◽  
Alexandra König ◽  
Sabrina Hebling ◽  
Christof R. Hauck ◽  
Matthias Frosch ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neisseria Meningitidis ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cell Types ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Src Inhibitor ◽  
Intracellular Signals

ABSTRACT Neisseria meningitidis, the causative agent of meningitis and septicemia, is able to attach to and invade a variety of cell types. In a previous study we showed that entry of N. meningitidis into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to α5β1-integrins. This interaction results in cytoskeletal remodeling and uptake of the bacteria. In this study we identified and characterized the intracellular signals involved in integrin-initiated uptake of N. meningitidis. We determined that the Src protein tyrosine kinases (PTKs) are activated in response to contact with N. meningitidis. Inhibition of Src PTK activity by the general tyrosine kinase inhibitor genistein and the specific Src inhibitor PP2 reduced Opc-mediated invasion of HBMEC and human embryonic kidney (HEK) 293T cells up to 90%. Moreover, overexpression of the cellular Src antagonist C-terminal Src kinase (CSK) also significantly reduced N. meningitidis invasion. Src PTK-deficient fibroblasts were impaired in the ability to internalize N. meningitidis and showed reduced phosphorylation of the cytoskeleton and decreased development of stress fibers. These data indicate that the Src family PTKs, particularly the Src protein, along with other proteins, are important signal proteins that are responsible for the transfer of signals from activated integrins to the cytoskeleton and thus mediate the endocytosis of N. meningitidis into brain endothelial cells.

scholarly journals Involvement of tyrosine kinases in the induction of cyclo-oxygenase-2 in human endothelial cells

1995 ◽  
Vol 312 (2) ◽  
pp. 419-423 ◽  
Author(s):  
A Blanco ◽  
A Habib ◽  
S Levy-Toledano ◽  
J Maclouf
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Interleukin 1 ◽  
Broad Band ◽  
Selective Reduction ◽  
Protein Tyrosine Kinases ◽  
Human Endothelial Cells ◽  
Protein Tyrosine ◽  
Cox 2 ◽  
Cox 1

In addition to a constitutive cyclo-oxygenase (Cox-1), human endothelial cells also possess an inducible cyclo-oxygenase (Cox-2) which plays an important role in the regulation of the synthesis of prostacyclin (prostaglandin I2). Cox-2 is regulated and expressed in large quantities upon activation of the cells by inducers such as phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), or interleukin-1 alpha. We have investigated the involvement of protein tyrosine kinases in Cox-2 expression by human endothelial cells upon activation by these inducers. PMA or interleukin-1 alpha provoke an increase in the phosphorylation of substrates of 110 and 120 kDa and additional phosphorylations for a broad band of multiple substrates in the 70 kDa range. This stimulation was accompanied by the induction of Cox-2 protein, detectable after stimulation for 1 h, which is consistent with an increase in activity reflected by prostacyclin synthesis; no variation in the expression of Cox-1 could be observed. Three distinct inhibitors of protein tyrosine kinases, genistein, herbimycin or AG-213, reduced tyrosine phosphorylation of cell substrates, consistently with their pharmacological effects. Under these conditions, there was selective reduction of Cox-2 expression without modification of Cox-1. Regulation of Cox-2 induction is also dependent on the activation of PKC since Ro 31-8220 or PKC depletion by PMA prevented its induction. Our results suggest that within the time-frame of our experiments these effects on kinases are specific for Cox-2 rather than Cox-1.

scholarly journals Protein-tyrosine-kinase-dependent expression of cyclo-oxygenase-1 and -2 mRNAs in human endothelial cells

1997 ◽  
Vol 322 (2) ◽  
pp. 373-377 ◽  
Author(s):  
Kenzo HIRAI ◽  
Hiroshi TAKAYAMA ◽  
Kenjiro TOMO ◽  
Minoru OKUMA
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Umbilical Vein ◽  
Protein Tyrosine Phosphatases ◽  
Conclusive Evidence ◽  
Protein Tyrosine Kinases ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Synergistic Enhancement ◽  
Interleukin 1Α

Endothelial cells possess constitutive or inducible cyclo-oxygenase (COX) isoenzymes for prostacyclin production, but the mechanisms for their expression are largely unknown. We found that vanadate, an inhibitor of protein-tyrosine phosphatases, induced the expression of two COX isoenzyme mRNAs in human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. Vanadate also stimulated an increase in COX-2 protein levels, but did not affect significantly the levels of constitutively expressed COX-1 protein. Synergistic enhancement of expression of the two COX isoenzyme mRNAs was observed on stimulation of HUVEC with vanadate plus interleukin-1α. Tyrphostin-47, which as an inhibitor of protein-tyrosine kinases abolished vanadate-induced protein-tyrosine phosphorylation, inhibited expression of the two COX isoenzyme mRNAs in HUVEC stimulated with vanadate or interleukin-1α. These data provide conclusive evidence that activation of protein-tyrosine kinases is causally linked to expression of the mRNAs for the two COX isoenzymes in HUVEC.

scholarly journals CGP 57148, a Tyrosine Kinase Inhibitor, Inhibits the Growth of Cells Expressing BCR-ABL, TEL-ABL, and TEL-PDGFR Fusion Proteins

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4947-4952 ◽  
Author(s):  
Martin Carroll ◽  
Sayuri Ohno-Jones ◽  
Shu Tamura ◽  
Elisabeth Buchdunger ◽  
Jürg Zimmermann ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine

Abstract CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

scholarly journals The SRC Kinase Family Member Lymphocyte Specific Kinase (p56Lck) Regulates Endothelial Cell Proliferation and Survival

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1445-1445
Author(s):  
Venkaiah Betapudi ◽  
Sergei M. Merkoulov ◽  
Suman Kundu ◽  
Ou Ji ◽  
Meifang Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tyrosine Kinases ◽  
Tube Formation ◽  
Protein Tyrosine Kinases ◽  
Endothelial Cell Proliferation ◽  
Protein Tyrosine ◽  
Lentiviral Transduction ◽  
Induction Of Apoptosis

Abstract Background and Objective: The Src family consists of Src, Lck (p56Lck), Fyn, Hck, Blk, Lyn, Fgr, Yes, and Yrk protein tyrosine kinases, which display cell and tissue specific expression. These non-receptor protein tyrosine kinases regulate many cellular processes including cell growth, differentiation, shape, migration, and survival. p56Lck is primarily expressed in T lymphocytes where it stimulates T cell receptor-mediated signaling to regulate thymocyte development. Similar to other SRC family members, p56Lck activity is negatively regulated by phosphorylation of Tyr505 and undergoes autophosphorylation on Tyr394 in the active state. Our previous studies showed that p56Lck activation occurred in proliferating endothelial cells exposed to cleaved high molecular weight kininogen (HKa), which induced endothelial cell apoptosis.The purpose of this study is to define the role of Lck in regulation of endothelial cell survival and angiogenesis. Methods: Primary cultures of endothelial cells were treated with Lck siRNA or with mammalian expression vectors or lentiviral constructs containing Lck cDNA. The proliferative capacity, viability, and tube-forming ability of Lck and control endothelial cells were determined. Results: Transfection of endothelial cells with Lck siRNA markedly changed their morphology and significantly enhanced their ability to proliferate in response to bFGF. In contrast, endothelial cells transfected with a Lck expression vector or transduced with a Lck-lentivirus not only failed to proliferate in response to growth factors, but also underwent apoptosis on several different extracellular matrix proteins. Finally, overexpression of Lck in endothelial cells led to impaired tube formation in a matrigel-based, in vitro angiogenesis assay; apoptosis of endothelial cells in this system was suggested by increased staining with Annexin V. Conclusion: Taken together, these results suggest that regulation of Lck activation provides a key node in determination of endothelial cell proliferation and survival that is likely to be relevant to the regulation of angiogenesis. Moreover, they suggest that specific endothelial cell Src family kinase members may play contrasting roles in regulation of these processes. Activation of Lck may represent a potential mechanism for controlling aberrant angiogenesis in pathological conditions. Figure: Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis. Figure:. Effect of control or p56Lck-expressing lentiviral transduction on endothelial cell tube formation and induction of apoptosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of Protein Tyrosine Kinases byCoxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

2001 ◽  
Vol 69 (4) ◽  
pp. 2520-2526 ◽  
Author(s):  
Sonia Meconi ◽  
Christian Capo ◽  
Maryse Remacle-Bonnet ◽  
Gilbert Pommier ◽  
Didier Raoult ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Q Fever ◽  
Protein Tyrosine Kinases ◽  
Actin Reorganization ◽  
Protein Tyrosine ◽  
Cytoskeleton Organization ◽  
Cytoskeleton Reorganization

ABSTRACT Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

scholarly journals Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.

1994 ◽  
Vol 14 (8) ◽  
pp. 5384-5391 ◽  
Author(s):  
W Rodgers ◽  
B Crise ◽  
J K Rose
Keyword(s):  
Tyrosine Kinases ◽  
Membrane Fraction ◽  
Protein Tyrosine Kinase ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Cell Types ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Glycosyl Phosphatidylinositol ◽  
Common Signal

Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.

scholarly journals Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases

2008 ◽  
Vol 76 (10) ◽  
pp. 4692-4702 ◽  
Author(s):  
Kaiping Deng ◽  
Jason R. Mock ◽  
Steven Greenberg ◽  
Nicolai S. C. van Oers ◽  
Eric J. Hansen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Immune Cell ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Site Directed Mutagenesis ◽  
Haemophilus Ducreyi ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine

ABSTRACTThe LspA proteins (LspA1 and LspA2) ofHaemophilus ducreyiare necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-typeH. ducreyicells were incubated with macrophages. LspA proteins in cell-free concentratedH. ducreyiculture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

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